The present invention relates generally to the field of neurological and physiological dysfunctions associated with Alzheimer""s Disease. More particularly, the invention is concerned with the identification of proteins associated with Alzheimer""s Disease, to methods of diagnosing Alzheimer""s Disease and to methods of screening for candidate compounds which modulate the interaction of certain proteins, specifically armadillo repeat proteins, with presenilin proteins.
Alzheimer""s Disease (AD) is a degenerative disorder of the human central nervous system characterized by progressive memory impairment and cognitive and intellectual decline during mid to late adult life (Katzman, 1986, N. Eng. J. Med. 314:964-973). The disease is accompanied by a constellation of neuropathologic features principal amongst which are the presence of extracellular amyloid or senile plaques, and neurofibrillary tangles in neurons. The etiology of this disease is complex, although in some families it appears to be inherited as an autosomal dominant trait. Genetic studies have identified three genes associated with the development of AD, namely: (1) xcex2-amyloid precursor protein (xcex2PAPP) (Chartier-Harlin et al., 1991, Nature 353:844-846; Goate et al., 1991, Nature 349:704-706; Murrell et al., 1991, Science 254:97-99; Karlinsky et al., 1992, Neurology 42:1445-1453; Mullan et al., 1992, Nature Genetics 1:345-347); (2) presenilin-1 (PS1) (Sherrington et al, 1995, Nature 375:754-760); and (3) presenilin-2 (PS2) (Rogaev et al., 1995, Nature 376:775-778; Levy-Lehad et al., 1995, Science 269:970-973).
The presenilin genes (presenilin 1xe2x80x94PS1 and presenilin 2xe2x80x94PS2) encode homologous polytopic transmembrane proteins that are expressed at low levels in intracellular membranes including the nuclear envelope, the endoplasmic reticulum, the Golgi apparatus and some as yet uncharacterised intracytoplasmic vesicles in many different cell types including neuronal and non-neuronal cells (Sherrington et al., 1995; Rogaev et al., 1995; Levy-Lahad et al., 1995; Doan et al., 1996, Neuron 17:1023-1030; Walter et al., 1996, Molec. Medicine 2:673-691; De Strooper et al., 1997, J. Biol. Chem 272:3590-3598; Lehmann et al., 1997, J.Biol.Chem. 272:12047-12051; Li et al., 1997, Cell 90:917-927). Structural studies predict that the presenilins contain between six and eight transmembrane (TM) domains organized such that the N-terminus, the C-terminus, and a large hydrophilic loop following the sixth TM domain are located in the cytoplasm or nucleoplasm, while the hydrophilic loop between TM1 and TM2 is located within the lumen of membranous intracellular organelles (Doan et al., 1996; De Strooper et al., 1997; Lehmann et al., 1997).
Missense mutations in the PS1 and PS2 genes are associated with the inherited forms of early-onset AD (Sherrington et al., 1995, Nature 375:754-760; Rogaev, et al., 1995, Nature 376:775-778; Levy-Lahad et al, 1995, Science 269: 970-973). Several lines of evidence have also suggested roles in developmental, apoptotic signalling and in the regulation of proteolytic cleavage of the b-amyloid precursor protein (bAPP) (Levitan et al., 1995, Nature 377:351-354; Wong et al., 1997, Nature 387:288-292; Shen et al., 1997, Cell 89:629-639; Wolozin et al., 1996, Science 274:1710-1713; De Strooper et al., 1998, Nature 391:387-390). Nevertheless, it remains unclear just how these putative functions are mediated, or how they relate to the abnormal metabolism of the xcex2APP associated with PS1 and PS2 mutations (Martin et al., 1995, NeuroReport 7:217-220; Scheuner et al., 1996, Nature Med. 2:864-870; Citron et al., 1997, Nature Med. 3:67-72; Duff et al., 1996, Nature 383:710-713; Borchelt et al., 1996, Neuron 17:1005-1013).
The identification and cloning of normal as well as mutant PS1 and PS2 genes and gene products are described in detail in copending commonly assigned U.S. application Ser. Nos. 08/431,048, filed Apr. 28, 1995; 08/496,841, filed Jun. 28, 1995; 08/509,359, filed Jul. 31, 1995; and 08/592,541, filed Jan. 26, 1996, the disclosures of which are incorporated herein by reference.
There is speculation that onset of AD may be associated with aberrant interactions between mutant presenilin proteins and normal forms of PS-interacting proteins, and these changes may increase or decrease interactions present with normal PS1, or cause interaction with a mutation-specific PS-interacting protein. Such aberrant interactions also may result from normal presenilins binding to mutant forms of the PS-interacting proteins. Therefore, mutations in the PS-interacting proteins may also be causative of AD.
While the identification of normal and mutant forms of PS proteins has greatly facilitated development of diagnostics and therapeutics, a need exists for new methods and reagents to more accurately and effectively diagnose and treat AD.
Applicants have discovered that both PS1 and PS2 interact specifically with at least two members of the armadillo family of proteins (GT24/Neuronal Plakoglobin Related Armadillo Protein and xcex2-catenin) that are expressed in both embryonic and post-natal tissues. Moreover, the domains of PS1 and PS2 that interact with these proteins have been identified. Applicants have also discovered that mutations in PS1 and PS2 affect the translocation of xcex2-catenin into the nucleus of both native cells and cells transfected with a mutant PS gene. These discoveries provide the basis for materials and methods useful in the diagnosis and treatment of AD.
One object of the invention is directed to a method for identifying substances that alter the interaction of a presenilin protein with a presenilin-binding protein, comprising:
(a) contacting at least the interacting domain of a presenilin protein to a presenilin-binding protein in the presence of a test substance, and
(b) measuring the interaction of the presenilin protein and the presenilin-binding protein. Preferably, the interacting domain is contained in or contains the sequence of amino acid residues from about 260 to about 409 of a mutant PS1 protein, more preferably the sequence of amino residues from about 372 to about 399, in which the amino acid positions correspond to the wild-type human PS1 sequence defined by SEQ ID NO:1. When PS2 is used, the sequence of amino acid residues from about 266 to about 390 are preferred, more preferably the sequence of amino residues from about 350 to about 380, in which the amino acid positions correspond to the wild-type human PS2 sequence defined by SEQ ID NO:2.
Substances identified that alter the interaction of a mutant presenilin protein with a normal presenilin-interacting protein, as well as the interaction of a normal presenilin protein with a mutant presenilin-interacting protein, are putative candidates for use in the diagnosis and treatment of AD.
Another object of the invention is to provide methods of identifying substances that modulate the nuclear translocation of an armadillo protein, comprising:
(a) providing a culture of cells that express the armadillo protein and a mutant presenilin protein, or a functional fragment thereof that binds said armadillo protein;
(b) contacting said culture with a test substance;
(c) inducing nuclear translocation of said armadillo protein in said cells; and
(d) measuring levels of nuclear armadillo protein as compared to a control as an indication of modulatory activity of said test substance. Alternatively, step (d) may comprise the step of measuring the effects of altered nuclear translocation such as: (i) alterations in transcription of downstream genes such as xcex2APP, xcex3-secretase, or by alteration in the activity of a transcription reporter assay such as the Tcf/Lef-luciferase assay; (ii) alterations in cellular responsiveness to signalling molecules (e.g., Wnt, EGF) which use armadillo proteins for intracellular signal transduction; or (iii) alterations in cell:cell adhesion (e.g., synapse formation) mediated by cytoplasmic armadillo proteins.
Armadillo proteins of the present invention include, but are not limited to, hNPRAP, p0071 and xcex2-catenin. Cells may be native or recombinant (i.e., the mutant PS gene and/or the armadillo protein gene are/is transgenic to the cell).
It is another object of the invention to provide methods for screening for carriers of presenilin alleles associated with AD or related disorders, comprising:
(a) obtaining cells from an individual to be tested for Alzheimer""s Disease or a related disorder;
(b) culturing said cells with a substance which induces nuclear translocation of an armadillo protein; and
(c) measuring levels of nuclear armadillo protein as compared to a control as an indication of the presence or absence of presenilin alleles associated with Alzheimer""s Disease or a related disorder. Alternatively, step (c) may comprise measuring effects of altered nuclear translocation such as: (i) alterations in transcription of downstream genes such as xcex2APP, xcex3-secretase, or by alteration in the activity of a transcription reporter assay such as the Tcf/Lef-luciferase assay; (ii) alterations in cellular responsiveness to signalling molecules (e.g., Wnt, EGF) which use armadillo proteins for intracellular signal transduction; or (iii) alteration in cell:cell adhesion (e.g., synapse formation) mediated by cytoplasmic armadillo proteins.
Inducement of nuclear translocation of the armadillo protein is preferably performed by activating the Wnt/armadillo signal transduction pathway of the cells. Most preferably, activation is with a lithium salt (e.g., lithium chloride) or with methods such as with recombinant Wnt proteins (or invertebrate homologues, e.g., Wingless proteins) applied exogenously to the medium or via transfection into the test cells.